Purified antidiabetic product and process of making it



Westerns-W f UNITED T PATENT -oi=rics;.

GEORGE B. WALD, OF FRANKLIN, INDIANA,- ASSIGNOR, BY. MESNE ASSIGNMENTS,

rowan-commons on-mnn UNIVERSITY or Ton-curd, or 'ronon'ro, ours-mo,

camera.

PUBIFIED ANTINIANETIC IRODUC'I AND PROCESS OF MAKING IT.

No Drawing. Continuation of application Serial No. 622,448, filed March 2, 1923. This application filed June 11. 1924.

'in stable and very pure. form the active.

anti-diabetic principle or hormone normally secreted by thepancreas of animals; by

isolating it much more completely than has heretofore been possible from contaminating and deleterious substances, largely .nitrogenous, with which it is associated in the pancreas and from which it has been impossible prior to my invention to separate it with anything like the completeness which I now attain. I

Moreover, it is the object of my invention to provide a suitable method of extraction and an eflic'ient method of purification, by which an anti-diabetic substance having the physiological and therapeutic characteristics and reactions of the active anti-diabetic principle or hormone of the pancreas may be obtained in purified and stable form, and substantially free of the substances with which it was originally associated in the pancreas; which method comprises treating the pancreas to leave behind the enzymes of the pancreas or inhibit their destructive action separating the active. anti-diabetic principle-,free of the major part of the substances,"la1'gely nitrogenous, with which it was originally associated, and then purifying this active anti-diabetic principle by reducing the residual-nitrogen content to apoint where it is not in excess of 0.1 milligrams per unit of anti-diabetic activity.

it has long been believed that the pancreas, including the pancreas of land animals and cartilaginous fishes, and the related glands. (principal islets) of bony fishes,-normall'y produces, as an internal Serial No. 719,269.

secretion, an active principle or hormone which is essential to the metabolism of su ar in the animal body, and that diabetes me litus ensues when this secretion fails or becomes insufiicient. Investigators sought in vainfor years to derive this active principle or hormone from the pancreas in a form that could-be administered with safety and would be efiective in relieving the diabetic syndrome when used for human ad ministration, but it was not until the work of Banting, Best and Collip, done at the University of Toronto, that such a product was derived, as set forth in their Patent No. 1,469,994, granted October 9, 1923.

Very briefly their process involves the following. maln steps 1. The extraction of the ground pancreas,-'before any considerable. quantity of the anti-diabetic hormone therein has been destroyed by trypsin and other enzymes of the pancreas,with a suitable solvent that extracts such hormone and either leaves behind the destructive enzymes such as trypsin or inhibits their destructive action on the anti-diabetic hormone, or both;

2. The Variation of the solvent-concentration of the solution, either with the original solvent or a different one or both, in order to separate by fractional precipitation and proper filtration, various components originally extracted. Alcohol has been successfully usedfor the fractional precipitation; and the precipitate which is obtained between and alcohol concentration, after the previous removal of precipitates obtained on lower alcohol concentration, contains at least part of the desired anti-diabetic hormone;

3. The solution in water of this hormonecontaining precipitate, which was obtained on 80% to 90% alcohol concentration; and 4. The standardization of the anti-dia betic activity of the solution thus obtained. Although other units of activity may be used, the unit of activity hereinafter referred to as a basis of calculation is that amount which when injected into a normal rabbit weighing 1 kg. reduces the blood sugar to below 0.045% within six hours and produces convulsions.

To my knowledge, the best product ob-- al-nitrogen content, under certain conditions,

caused unpleasant efiects, such as sensitization and induration at the point of injection. The contaminating substances present, also included :-substances containing phosphorus, substances containing purine groups, and substances containing pyrimidine grou s.

The resldual-nitrogen content of the antidiabetic product obtained by my invention is greatly reduced in comparison to that of previous products. It is-not more than 0.1

milligram per unit of anti-diabetic activity, and is usually of the order of 0.005 to 0.05 milligram per unit of anti-diabetic activity; whereas no previous product of which I am aware has had a residual-nitroigen content as low as 0.1 milligram per unit of anti-diabetic activity. To obtain this degree of purity, several repeated processes of purification may sometimes be required. Probably at least some of this small amount of residual nitrogen is in thehormone itself, and is not in a contaminating substance; so that the resultant advantage in the rduction of nitrogenous contamination is in even greater proportion than these figures show. In addition, the product obtained according to my invention is substantially free from water-soluble impurities :such as inorganic salts, substances containing phosphorus, substances containing purine groups, and substances containing pyrimidine groups,-which are noticeably present in the product which was obtained by the' previously known methods.

The final solution of the product obtained according to my invention is brilliantly clear, and in small quantities is practically as colorless as water, though in bulk it shows a light straw-color tinge; whereas previous products showed considerable color even in small quantities.

Thus it is possible to obtain a product which can be administered with maximum safety and maximum predetermination of its anti-diabetic effect. This product has a stability many times as great and a purity ranging from ten to one hundred times as great as the best product obtainable prior to my invention. This stability is so great that the product shows no appreciable diminution in potency after a lapse of over three months from the time of its preparation; andthispurity is so great'that out of many thousands of patients to whom the product has so far been administered, in-

cluding some who exhibited sensitization and induration effects with the best products previously obtainable, to my knowledge not a single instance of sensitization or induration or any other deleterious effect from the product has been reported.

By reason of this great purity, the effect obtained .on administration of the product is limited very closely if not wholly to the re lief of the diabetic syndrome, without material undesirable unrelated efiectssuch as 'indurati-on and sensitization.

' trogenous, so that they do not interfere with the potency or stability of the anti-diabetic substance and so that the residual-nitrogen content is not in excess of 0.1 milligram per unit of anti-diabetic activity.

I have discovered that the stability of the solution heretofore regarded as the final product can be controlled, and that it is affected at least in part, by the hydrogen ion concentration of such solution. lffthe hydrogen ion concentration is within a P range of from about 4 to 7 a deterioration of the anti-diabetic potency of the solution occurs at a rate dependent at least in part on the hydrogen ion concentration. Possi- 'bly this deterioration-is in part due-to a destructive action of contaminating nitrogenous matter present in the solution; but I have found that at least a large part of it is not the result of destruction of the hormone, but is the result of the slow formation from the solution of a precipitate containing the anti-diabetic hormone, thereby reducing the activityv of the solution. The rate and extent of formation of this hormone-containing precipitate vary with the hydrogen ion concentration of the solution, and they increase as its isoelectric point is approached from either side. "(The term isoelectric point is here used toindicate the P at which certain'substances in so lution tend most strongly to come o ut'of solution. The isoelectric point varies with different substances.) The lower the concentration of impurities in the solution,'the more clearly delimited is this isoelectric point, the more rapid is the rate of forma- 1 tration of the impurities in assume the yield of the precipitate. Thiaprecigr tate starts to form, under favorable con tions,'at-a P of about 4, reaches'almaxh mum at a P of about 4.5 to 5.5,and is sub-' stantially completelfi edissolved at a' P of,about'6.5 to 7. owever, if the concenso hfgh that the precipitate, which would otherwise form, ,is

will form. I In carrying out necessary but it is. advantageous to take a hydrogen ion concentration,

product such as that finally obtained by'previous processes, and though purification of such a product can be effected by various methods, I prefer to do it by adjusting the of a. solution containing the hormone. to the vicinity o an isoelectric point which resultsin the separation of a precipitate containing .such hormone or having such hormone associated with. it. As one way of separating and obtaining the stable .and pure anti-diabetic substance, this method of isoelectric precipitation of a substance containing the anti-dia betic principle or hormone makes profitable use of the very precipitate-forming tendency that previously caused trouble.

Although this method of isoelectric precipitation maybe applied with good effect to solutions obtained earlier than the final one of previous processes, it is preferred to use it on solutions of previous final products, from which the great mass of contaminating matter has been removed by such prior processes; because the effectiveness of this meth od increases with the freedom of the hormone from associated contaminating substances in the solution from which the precipitation is made.

When using this method of isoelectric precipitation, I deliberately adjust the hydrogen ion concentration of the solution containing the anti-diabetic substance to the isoelectric point of the latter in order to precipitate it in very pure and very stable form and practically free of the contaminating substances, largely nitrogenous, with which it was associated in the solution. In other words, the hydrogen ion concentration is adjusted as nearly as possible to the isoelectric point of the substance or substances, which on precipitation from such solution include or have associated with them the anti-diabetic hormone, whether or not such substance or substances constitute such hormone. In this. specification, and in some of the claims, the term anti-diabetic substance. is used to denote generically the antidiabetic hormone whether alone or in association with something else. The solution or anti-diabetic substance from which the isoelectric precipitation is made can be the solution is" may held in solution, the, 80- lution' can be diluted so-that the precipitate -e I I -seems to be somewhere between 4.5 and 5.5 my invention, it'is not f with a P between 4.5 and ca'usedto beatthe the prior process by but if the P3 is too addin an alkali,

which it was obtained; low it can be raised by preferably sodium hydroxi e, until the vicinit [of the isoelectric point is reached, while i it is too hi h an acid can be added preferably hydroc oric acid. As the precipitate forms a variation occurin the hydrogen ion concentration, but it is not necessary tomaintain a P for while the isoelectric point on the P scale, or in other words P 530.5, as nearly as I have been able to determine, the precipitate is obtained to a greater or desired P}; by controlling less extent through the P range 4 to 7, so

that a hydrogen ion concentration at any point within this range can be used. The best results I have .so far obtained have been 5.5. After the P has been adjusted as closely as possible to the isoelectric point, of the anti-diabetic substance, the solution is allowed to stand to permit the formation of the precipitate. Several days or a week are .preferably allowed for this, though a separable precipitate is obtained within a few hours. If the precipitate is slow informing, or does not form at all, the solution is diluted, maintaining such hydrogen ion concentration as is necessary to makethe precipitate appear. At intervals the precipitate is removed, as by filtration; tration is preferably readjusted to the optimum P above mentioned whereby further amounts of the precipitate are obtained. To accelerate the formation of this precipitate, it is advantageous to chillthe solution during this period of standing.

The recipitates th s obtained are dislower .if desired, and it is preferred to use a P as low as 2, at which P the amount of acid is not sufiicient to roduce undue stinging in administration, if hydrochloric acid is used. For special purposes, a higher P may beused, either within such isoelectric range or above it. Solutions with a P as high as 12 have been effectively used.

The final solutions I obtain are very stable and very pure. Apparently this great puri-. fication is due to the and the hydrogen ion concenfact that the final pre and it has been found of 3.5 is suitable for ordinary therapeutic purposes; but the P may be cipitate of .ianti-diabetic substance contains only the hormone and some very closely associated or combined substance or substances having no appreciable inju'rious solution before the precipitation.

effect on the stabi1ity,-potency,. or anti-diabetic properties of the final product, and that the major part of the contaminating substances (proteins, split protein products, other nitrogenous substances, inorganic salts, etc.) remain in the solution and are separated from .the anti-diabetic product.

The .total number of units of potent antidiabetic substance recovered by this method is sometimes as high' as 90% of those in the This varies with the completeness of the precipitation obtained and is increased ;by having a purer. starting product; by increasing the standing period; by reducing the temperature during standing; and especially by getting closer to the isoelectric point. The loss in anti-diabetidactivity may be made comparatively slight by exercising care on these points; and in any case it is far outweighed by the manifold increase obtained in the stability and purity of the product.

I am aware that Banting, 'Best, and

Collip used the principle of isoelectric pre-' cipitation in their work at Toronto. But they used it for different purposes and with I different effects. They used it with the idea tion and throwing away the of precipitating undesirables, and of leaving the anti-diabetic hormone .inthe soluprecipitate obtained. On the other hand, I use it to precipitate and conserve the anti-diabetic prodnot, and leave the undesirables in the solution; and I discard thesolution whenall of the anti-diabetic hormone has been separated from it, and preserve the precipitate.

Having thus fully described my invention, what I claim as new and desire to secure b Letters Patent is 1. T e method of obtaining an active anti-diabetic substance, having the physiological and therapeutic characteristics and reactions of the anti-diabetic principle or hormone of the pancreas, in purified and stable form, and substantially free of the subtstances with which it was originally associated .in the pancreas, which method comprises treating the pancreas to leave behind the enzymes of the pancreas or inhibit their destructive action, separating the active anti-diabetic principle free of the major part of the substances, largely nitrogenous. with Which it was. originally associated, and then purifying this active antidiabetic-principle by reducing the residualnitrogen content to a point where it is not in excess of 0.1 milligram per unit of antidiab'etic activity.

53. The method of obtaining the active anti-diabetic principle or hormone of the 7 pancreas substantially free of substances with which it was originally associated in the pancreas, which method comprises sepa;

rating as a crude product the active antidiabetic principle or hormone free from the major part of such. substances, and then separating and obtaining the active ,antidiabetic principle or hormone free ,from all.

residual nitrogen in excess of 0.1 milligram per unit of-anti-diabetic activity.

3. The method of obtaining the active anti-diabetic princi le orv hormone. of the pancreas substantia 1y free of substances with which it was originally associated in the pancreas as claimed in claim. 2, and making an aqueous solution of the antidiabetic product that retains its anti v -separate the active anti-diabetic principle or' hormone from the major part of the contaminating substances, forming a solution containing said separated hormone and causing to be formed in' the solution a precipitate, with not more residual nitrogen than" 0.1 milligram per unit of anti diabetic activity, which precipitate. includes orehas associated with it the active antidiabetic principle or hormone whether or not such precipitate constitutes suchhor- 5. The method, as claimed in claim-4,

with the added step of making a solution of the precipitate formed, which precipitate includes or has associated with it the'active anti-diabetic principle or hormone.

H 6. Aprocess of preparing an anti-diabetic product from the pancreas, which process consists in treating the pancreas to obtain the anti-diabetic principle or hormone and leave behind .the enzymes of the pancreas or inhibit their destructive action, separating the anti-diabetic principle or hormone from the major part of the pancreas-residue.- forming of the product containing the said anti-diabetic principle or hormone a solu tion containing said hormone and the then co-pre'sent matter and adjusting the hydrogen ion concentration of said solution to the vicinity of the isoelectric point'of a substance, which, following upon the sa-id adjustment, forms a precipitate including the anti-diabetic, hormone, and separating and preserving the precipitate thus formed.

a point betic product from diabetic principle or residue and treating principle or hormone Substantially free from the major part of the substances with which it was associated in the pancreas, and then adjusting the hydrogen ion concentration of such solution to the vicinity of the isoelectric such adjustment, forms a precipitate including the active anti-diabetic princi 1e or hormone, and separating from the liquid and preserving the precipitate thus formed. v

8. A process of separating the active antidiabetic principle or hormone of the pancreas from contaminating siibstances with which it was originally associated in the pancreas, which process comprises adjusting the hydrogen ion concentration of a solution containing such anti-diabetic principle or hormone to the vicinity point of a substance which, following upon the'said adjustment, forms a precipitate including the anti-diabetic principle or. hormone, and separating from-the liquid and preserving the precipitate thus formed.

9. A process of purifying the product containing the active anti-diabetic principle or hormone of the ancreas as set forth in claim 8, with the ded step of :forming of the separated precipitate so obtained a solution having a hydrogen ion concentration sufliciently far from such isoelectric point to prevent material recipitation.

10. A process 0? purifying a product containing the anti-diabetic hormone of the pancreas as set forth in claim 8, with the added step of forming of the separated precipitate so obtained a solution having a hydrogen ion concentration which is sufficiently on the acid side of such isoelectric point to prevent material precipitation.

'11. A process of obtaining an anti-diathe pancreas substantially free of contaminating substances with which it wasoriginally associated, which process comprises extracting the pancreas with a solvent that takes'up the active antihormone without destroying it or inhibits its destruction by enzymes coresent with it in the'pancreas, separating t e solution from the pancreasthis solution to separate said hormone from the major part of the contaminating substances, producing further separation of said contaminatingsubstances by fractional precipitation at varying concentrations of alcohol, and separating the fraction obtained between and.90% alcohol concentration, forming asoIution of said last-named fraction and adjusting the hydrogen ion concentration of said last-named solution to the of a substance which, following upon pancreas, separating of the isoelectric.

betic product from the pancreas,

hormone from,

'sa dsolut1on vicinity of the isoelectric point of a substance which, following upon the said justment, forms a precipitate including the anti-diabetic hormone, and separating and preserving the precipitate thus formed.

12. A process of preparing an anti-diabetic product from the pancreas, 'which process consists in eirtracting the pancreaswith a solvent that takes up the anti-diabetic hormone thereof without destroying it or prevents its destruction by enzymes such as trypsin co-present wit it -in the the solution from the pancreas-residue, producing separation of said hormone from contaminating substances by fractional'preci itation at varying concentrations of alco ol! and-preserving the precipitate obtained between 80% and 90% alcohol concentration, forming a solution of saidlast-named precipitate and adjusting the hydrogen ion concentration of the said last-named solution'to the vicinity of the isoelectric pointof asubstance which, following upon the said adjustment, forms a precipitate including the anti-diabetic hormone, and separatin and preserving thet precipitate thus forme i 13. A process of preparing an anti-diawhich in extracting the pancreas process consists that takes up the anti-diawith a solvent betic hormone thereof without destroying 1t 0r prevents its destruction by enzymes suc as trypsin co-present with it in the pancreas, separating the solution from the pancreas-residue and treating it to separate said hormone from taminating substances, forming a solution containing said separated hormone and the then co-present matter and adjusting the last hydrogen ion concentration. of said named solution tothe vicinity of the isoelectrio point of a substance which, following upon the said adjustment, forms aiprecipitate including the anti-diabetic hormone, and separating and tate thus formed.

Q 14. A process of, preparing an anti-diabetic .product from the pancreas, which process consists in extracting the pancreas with a solvent that takes-up the anti-diabetic principle or hormone thereof without destroying it orprevents its destruction by enzymes such as'trypsm co-present with it in the pancreas, obtaining separate from the pancreas-residue a solution containing said hormone and the then co-present matter and adjusting the hydrogen ion concentration of to the vicinity of theisoelectrio point of a substance, which, .following upon the said adjustment, forms a precipitate including the anti-diabetic hormone, and separating and preserving the precipi-Z tate thus formed.

15. A method of purifying" the. active the major part of the con- 5 preserving the precipianti-diabetic substance of the containing the anti-diabetic hormone of the b pancreas whilefin solution, which method comprises adjusting the hydrogen ion concentration of the solution to thevicinity of-the isoelectr'ic pointof said substance. in order'to precipitate it, and then separating it from the solution and preserving it. 1

16. A process of separating a substance containing the anti-diabetic hormone of the pancreas from contaminating substances, which process consists in adjusting the hydrogen ion'concentration of a solution of a pancreas-derived substance containing said hormone and other matter to a P between 4 and 7 to produce a precipitate which includes said hormone but leaves behind in the solution the major part of the contaminating nitrogenous matter and inorganic salts, and separating this precipitate from the solution and preserving it.

17. A process of se aratinga substancecontaining the anti-dia etic hormone of the pancreas from contaminating substances,

which process consists in adjusting the hyv drogen io concentration of a solution of a 'pancreaserived substance containing said hormone and other matter to a P between 4' and 7 to produce 'aprecipitate which includes said hormone but leaves behind in the solution the major ,part of the contaminating nitrogenous salts, separating this precipitate from the solution, and formin of this precipitate an aqueous solution with. a. hydrogen ion concentration below P 4. r

.18. A process of separating a substance pancreas from contaminating substances, which process consists in adjusting the hydrogen ion concentration of a solution ofa pancreas-derived substance containing said hormone and other matter to a P between 4 and 7 to produce a recipitate which includes said hormone ut leaves behind in' the solution the major part of the contaminating ni'tro enous matter and inorganic salts, separating this precipitate from the b of this precipitate an a hydrogen ion concentration outside the P range from 4 to 7.

19. A process of purifying the active anti-diabetic substance of the pancreas,

solution, and forming aqueous solution wit which process consists in adjusting the by- -major part of contaminating drogeu ion concentration of a solution of a substance derived from. the pancreas and containing said active anti-diabetic principle or hormone and other matter to P 5i0.5 to produce a precipitate which includes said anti-diabetic principle or hormone but leaves behind in the solution the nitrogenous matter. and inorganic salts, and separating this precipitate from the solution and preserving it.

20. A process of purifying the active 1 tivity,

matter and inorganic N anti-diabetic substance of the pancreas as set forth in claim 19 with the added step of forming of the final with a hydrogen :ion concentration on the acid side of P 4.

,21. An anti-diabetic product derived from the pancreas, which product has a residual-nitrogen content of not more than 0.1 milligram per unit of anti-diabetic acand upon hypodermic administration relieves the diabetic syndrome. 22. An anti-diabetic from the pancreas, which product has a residual-nitrogen content of the order of 0.005 to 0.05 milligrams per unit of antidiabetic activity, and upon hypodermic administration relieves the diabetic syndrome' upon hypodermic administration relieves the diabetic syndrome. a

25. A solution of an anti-diabetic product derived from the pancreas and containing the anti-diabetic principle or hormone of the pancreas, dermica lly administered relieves the diaetic syndrome, is sufliciently free from injurioussubstances for repeated administration, and has an anti-diabetic potency which remains-substantially constant for at least three months from the time of its preparation.

26. An anti-diabetic product which contains the anti-diabetic principle or hormone of the pancreas, which product when hypodermically administered relieves'the diaetic syndrome without material unrelated effects. is pr'ecipitable from water solution at an isoelectric point in" the P range 4.5 to 5.5, and has a residual-nitrogen content of not more than 0.1 milligrams per unit of anti-diabetic activity. I i

27. An anti-diabetic product 7 from the pancreas and containing the active anti-diabetic principle or hormone of the pancreas, which product is substantially free from one or more of the following: substances containing phosphorus," substances containing purine groups, substances containing pyrimidine groups.

28. An anti-diabetic product containing the active anti-diabetic principle or hormone of the pancreas, which product has an antidiabetic potency that remains substantially constant for at least three months product derived I which solution when hypo derived precipitate a solution .from the time of its preparation and when the orderof 0.005 to 0.05 milligram per administered relieves the diabetic syndrome. unit of anti-diabetic activity. I 29. A substance as claimed in claim 28, in In witness whereof, I have hereunto set which the residual-nitrogen content is not my hand at Indianapolis, Indiana, this 9th 6 more than 0.1 milligram per unit of antiday of June, A. 1)."0116 thousand nine hundiabetic activity. dred and twenty four. 30. A substance as claimed in claim 28, in which the residual-nitrogen content is of GEORGE B. WALDEN. 

